The mammalian shcA gene encodes three overlapping proteins of 46, 52 and 66 kDa, that differ only in the extent of their N-terminal sequences (1). These shcA gene products share a C-terminal Src homology 2 (SH2) domain, a central proline-rich region (CH1), and a more N-terminal phosphotyrosine-binding (PTB) domain (2, 3). The ShcA SH2 domain binds preferentially to phosphotyrosine sites with the sequence pTyr-(hydrophobic/Glu)-X-(Ile/Leu/Met) [SEQ ID NO:9], and recognizes specific autophosphorylation sites in the activated epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-receptors (4, 5, 6). In contrast, the ShcA PTB domain has recently been shown to bind with high affinity to phosphotyrosine sites with the consensus sequence Leu/Ile-X-Asn-Pro-X-pTyr [SEQ ID NO:10]. Such sites are found in a number of growth factor receptors, notably the nerve growth factor receptor (Trk), and in polyoma middle T antigen (7, 8). The ShcA PTB domain, therefore, recognizes phosphotyrosine in the context of amino-terminal residues, as distinct from SH2 domains, which recognize amino acids C-terminal to phosphotyrosine (9). The p66 ShcA isoform, which is generated by alternative splicing, has an additional proline-rich N-terminal sequence (CH2) (Migliaccio, et. al).
These results have indicated that ShcA proteins have two modules that bind phosphotyrosine sites with entirely different specificities. Potentially for this reason, ShcA is a prominent substrate for tyrosine phosphorylation in cells stimulated with a wide variety of growth factors and cytokines, and in lymphoid cells stimulated with antigen (10, 11, 12, 13, 14). In addition, ShcA proteins are phosphorylated by oncogenic receptor and cytoplasmic tyrosine kinases (15, 16).
The principal phosphorylation site of human ShcA phosphorylation is at Tyr 317, located in the central CH1 region within the motif Tyr-Val-Asn-Val (aa 317-320 of SEQ ID NO:7, 17). A very similar element is found in mouse ShcA (Tyr313-Val-Asn-Ile; aa 313-316 of SEQ ID NO:6). Phosphorylation of this residue creates a high affinity binding site for the SH2 domain of a second adaptor protein, Grb2, which binds preferentially to phosphotyrosine sites with Asn at the +2 position (4). Grb2 is, in turn, associated through its SH3 domains with proline-rich motifs in the C-terminal tail of mSos1, a Ras guanine nucleotide exchange factor. ShcA phosphorylation, therefore, induces the formation of a ternary complex containing ShcA, Grb2 and mSos1, which may activate the Ras pathway (9). Consistent with this possibility, ShcA overexpression induces transformation of rodent fibroblasts in a fashion that is dependent on Tyr 317 (17). ShcA overexpression also elicits Ras-dependent neurite outgrowth in PC12 neuronal cells (18). This latter observation suggests that the binding of autophosphorylated Trk to the ShcA PTB domain, and ensuing ShcA phosphorylation and association with Grb2, is one mechanism by which the Trk tyrosine kinase might activate the Ras pathway (19, 20).
The significance of ShcA in signal transduction has been underscored by the identification of a Drosophila Shc protein that interacts through its PTB domain with the activated Drosophila EGF-receptor (21). Analysis of Drosophila Shc has also raised the possibility that Shc proteins have functions in addition to Ras activation.